A vast array of specific metabolic, developmental, and catabolic processes appear to be directly or indirectly regulated in vivo by comparatively small molecules such as steroids, retinoids and thyroid hormones. The mechanism whereby a single such compound can contribute to the regulation of numerous different cellular events was the subject of much speculation until relatively recently, when it was discovered that these compounds each share the ability to bind to transcriptionally active proteinaceous receptors. These protein receptors, in turn, are able to bind specific cis-acting nucleic acid regulatory sequence regions, termed response elements or RE's, located upstream of the coding sequence of certain genes and to activate the transcription of these genes. Thus, the proteinaceous receptors can serve as specific, ligand-dependent regulators of gene transcription and expression.
The amino acid sequences of these various receptors were quickly found to share regions of homology, thus making each such receptor a member of a family of ligand-modulated receptor molecules. This family has been termed the steroid superfamily of nuclear hormone receptors; nuclear, because the receptors are usually found in high concentration in the nucleus of the cell.
Further study of the structural and functional relationship between the nuclear hormone receptors has shown certain characteristics in common between them in addition to sequence homology. See e.g., Evans et al. Science 240:889-895 (1988). As stated above, the nuclear hormone receptors are able to bind to cis-acting regulatory elements present in the promoters of the target genes. The glucocorticoid, estrogen, androgen, progestin, and mineralcorticoid receptors have been found to bind as homodimers to specific response elements organized as inverted repeats.
Another class of nuclear hormone receptors, which includes the retinoid receptor RAR (retinoic acid receptor), the thyroid receptor, the vitamin D receptor, the peroxisome proliferator receptor, and the insect ecdysone receptor bind their response element as a heterodimer in conjunction with the retinoid X receptor (RXR), which in turn is positively activated by 9-cis retinoic acid. See Mangelsdorf, et al., The Retinoid Receptors in The Retinoids: Biology, Chemistry and Medicine Ch.8 (Sporn et al., eds. 2d ed., Raven Press Ltd. 1994); Nagpal and Chandraratna, Current Pharm. Design 2:295-316 (1996), which are both incorporated by reference herein. The retinoid receptors RAR and RXR, like many nuclear receptors, exist as a number of subtypes (RARα, RARβ, RARγ, and RXRα, RXRβ, and RXRγ). Additionally, each subtype may exist in different isoforms.
While the nuclear hormone receptors referenced above have all been shown to have specific ligand partners, nucleic acid and amino acid sequencing experiments and sequence alignment and comparison have revealed a class of protein molecules retaining significant sequence homology and structural similarity to the nuclear hormone receptor superfamily, but for which no corresponding ligand has yet been discovered. In fact, some of these “receptors” have been discovered to require no ligand binding to exhibit transcriptional activity. These receptors have been collectively termed “orphan” receptors.
Products of intermediate metabolism are known transcriptional regulators in prokaryotes and lower eukaryotes such as yeast; thus there has been speculation that such metabolites may also serve this function in higher organisms, perhaps through interaction with the nuclear hormone receptors.
Farnesol is an isoprenoid involved in the mevalonate biosynthetic pathway, which leads to the synthesis of cholesterol, bile acids, porphyrin, dolichol, ubiquinone, carotenoids, retinoids, vitamin D, seroid hormones, and farnesylated proteins. Farnesyl pyrophosphate, a derivative of farnesol, is the last common intermediate in the mevalonate biosynthetic pathway.
Foreman et al., Cell 81:687-693 (1995) have demonstrated that an orphan receptor, now termed farnesoid X-activated receptor (FXR), is activated by farnesol and related molecules. This reference is hereby incorporated by reference herein. FXR is expressed in the liver, gut, adrenal gland, and kidney.
The amino acid sequence of FXR reveals a conserved DNA-binding domain (DBD) and ligand-binding domain (LBD). The LBD comprises subdomains responsible for ligand binding, receptor dimerization, and transactivation. Additionally, cells expressing chimeric proteins that contain the LBD of FXR fused to the DBD of the yeast GAL4 transcription activator did not transcribe a reporter gene containing a GAL4 response element unless the FXR construct was coexpressed with another protein comprising the dimerization and ligand binding subdomains of RXR. These data suggested that FXR and RXR interact to form a transcriptionally active dimer. No interaction was seen between FXR and any other nuclear hormone receptors that were tested. Id.
Among the nuclear hormone receptors amino acid sequence homology to FXR is high in the insect ecdysone receptor (EcR), which dimerizes with an RXR homolog. When dimerized with RXRα, FXR was shown to specifically bind hsp27, an EcR response element, however, binding was not seen when FXR was expressed alone. FXR and RXR bind to certain sequences as a heterodimer.
The FXR-RXRα complex was found to be activated by juvenile hormone III (JH III); incubation of cells transfected with RXR and FXR. The cells were also transfected with a reporter plasmid containing 5 copies of the hsp27 response element within a portion of the mouse mammary tumor virus (MTV) promoter; the promoter was positioned upstream of the firefly luciferase gene. Activation of this gene results in the expression of luciferase, which is easily quantifiable as a measure of transactivation activity. Other potential ligands, including selected steriods, and eicosanoids were found to have no effect in this system. JH III failed to activate other nuclear hormone receptors, and does not activate either FXR or RXR alone. Forman et al., Cell 81:687-693 (1995).
JH III is a derivative of farnesyl pyrophosphate. Other farnesyl derivatives have been tested for the ability to activate the FXR-RXR complex. Farnesol was demonstrated to strongly activate the heterodimer. Other derivatives such as farnesal, farnesyl acetate, farnesoic acid and geranylgeraniol activated the FXR-RXR complex somewhat less strongly; the farnesyl metabolites geraniol, squalene and cholesterol did not activate FXR-RXR. Id.
Cholesterol synthesis is closely regulated by modulation of the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG-CoA), which regulates the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate. Through a series of phosphorylations and a decarboxylation reaction, mevalonate is converted into 3-isopentenyl pyrophosphoric acid, which isomerizes to 3,3-dimethylallyl pyrophosphoric acid. An enzyme-mediated condensation reaction between the 5 carbon isoprenyl compounds 3-isopentenyl pyrophosphoric acid and 3,3-dimethylallyl pyrophosphoric acid results in the formation of the 10 carbon diisoprenyl compound geranyl pyrophosphoric acid. This, in turn, reacts with another molecule of 3-isopentenyl pyrophosphoric acid to form the 15 carbon compound farnesyl pyrophosphate. Two molecules of this latter compound react to form the 30 carbon molecule presqualine pyrophosphate, which is dephosphorylated to form squaline. Squaline is then cyclized to form cholesterol. Thus, HMG-CoA reductase mediates the initial formation of the isoprene units that are subsequently assembled in series and cyclized to form cholesterol.
The levels of HMG-CoA reductase are governed in part by controlling the gene transcription, translation, and by degradation of the enzyme. Farnesol has recently been shown to be involved in the regulation of HMG-CoA reductase degradation. Evidence exists for the synergistic promotion of HMG-CoA reductase degradation by farnesol and a sterol component, such as 25-hydroxycholesterol. See e.g., Meigs et al., J. Biol. Chem. 271:7916-7922 (1996), hereby incorporated by reference herein.
Cholesterol is the precursor of various compounds such as sterols, bile acids such as cholic acid, and the steroid hormones such as testosterone and progesterone. All these compounds retain the basic cholesterol nucleus. The more polar bile acids are formed in the liver and secreted into the small intestine, where they aid in the absorption of lipids. The formation of bile acids from cholesterol is therefore an important degradation pathway for cholesterol, and is a key determinant of the steady-state concentration of cholesterol in the body.
The rate-limiting enzyme in the formation of bile acids from cholesterol is cholesterol 7α-hydrolase (Cyp7a). For some time it has been known that bile acids act in a negative feedback loop to limit their own production via this pathway, but the means by which this is accomplished has remained elusive. Recently, there has been evidence that Cyp7a synthesis and expression is inhibited by bile acids. Chiang, Front. Biosci. 3:D176-93 (1998) hereby incorporated by reference herein.
Despite the fact that cholesterol is essential for the synthesis of cell membranes and various hormones and other small molecules, raised levels of cholesterol, particularly in the form of low density lipoprotein (LDL), have been strongly linked to arteriosclerosis and other cardiovascular diseases. Additionally, maintenance of appropriate bile acid concentrations is important in regulating lipid metabolism, and may be useful in the prevention of colon cancer and gallstone formation.
Among currently available drugs for the treatment of hypercholesterolemia are ion exchange media such as colestipol and cholestyramine. These drugs function by sequestering bile acids in the gut; the bile acids are then excreted in the feces. Because the intestine does not reabsorb the sequestered bile acids, the bile acids are no longer available to inhibit the formation of bile acids by cholesterol degradation. As a result, bile acid synthesis is “depressed” with the result that the steady-state concentration of cholesterol is lowered.
Unfortunately, these ion exchange drugs have been associated with an increased incidence of intestinal tumors in rodents. Additionally, since the drugs are highly charged, they are capable of adsorbing other compounds, such as ingested drugs, naturally occurring hormones, regulatory factors and the like.
Recently a poster displayed by Neisor, Flach, Weinberger & Bentzen at an AACR conference on Nuclear Receptors in Palm Springs, Calif. held on Jan. 8-11, 1999 discussed the ability of certain 1,1-biphosphonate esters to activate FXR and to lower plasma cholesterol levels in mammals. This poster abstract is incorporated by reference herein.
Thus, there remains a need in the art for methods of modulating the steady-state concentration of cholesterol and/or bile acids. Such methods preferably do not significantly interact with other therapeutic agents, and function to help promote the breakdown or formation of cholesterol in a more direct fashion.